When killer lymphocytes recognize cells infected with intracellular pathogens, they release their cytotoxic granule contents to induce apoptosis of the infected target cell. However, what happens to intracellular parasites during this process is unclear. Host cell apoptosis is triggered by cytotoxic granule proteases (granzymes, Gzm), delivered into the target cell by the cholesterol-dependent membrane perturbing protein, perforin (PFN). Cytotoxic granules of humans and some other mammals, but not rodents, contain another pore-forming protein, granulysin (GNLY), which preferentially disrupts cholesterol-poor bacterial, fungal and parasite membranes. We recently found that when human killer lymphocytes recognize bacteria-infected cells, they rapidly kill intracellular bacteria. GNLY delivers Gzms into bacteria where they proteolytically attack bacterial electron transport chain complex I to generate toxic superoxide anion and also proteolytically destroy bacterial oxidative defense enzymes. Preliminary data show that GNLY also delivers Gzms into protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii, Leishmania major), which are rapidly killed, also in a superoxide-dependent manner. Destruction of intracellular parasites relies on the concerted action of all three cytotoxic effector molecules - Gzms, PFN and GNLY. Mice that express a GNLY transgene in killer lymphocytes are able to survive a T. cruzi challenge that is lethal to wild-type (WT) mice. This proposal uses T. cruzi as a model to investigate the hypothesis that GNLY, Gzm and PFN-mediated killing of intracellular protozoan parasites provides strong immune protection against intracellular parasites. We hypothesize that T. cruzi amastigotes are killed before the host cell, which limits the spread of infection. We also hypothesize that killer cells rapidly induce programs of cell death in T. cruzi that share some features with death pathways activated by Gzms in mammalian cells or bacteria. We also hypothesize that direct parasite killing helps control acute infection, enhance vaccine protection and reduce the chagasic pathology of chronic infection. This proposal joins the expertise of two principal investigators, who are leaders in studying (1) how killer cells destroy infected host cels and their pathogens and (2) the immune response to T. cruzi. The aims of the proposal are to define the features of killer cell-mediated parasite death, identify T. cruzi Gzm substrates that are important in causing death and explore how direct parasite killing contributes to immune defense by comparing infection in WT mice that do not express GNLY with GNLY-transgenic mice.